Neuroglobin (Ngb) is a recently discovered tissue globin with a high affinity for oxygen expressed in the vertebrate brain. Initial observations suggest that Ngb is neuroprotective against hypoxic-ischemicinsults. However, the underlying neuroprotective mechanisms remain to be defined. We propose 3 aims to investigate the overall hypothesis that under hypoxia/ischemia conditions, elevated Ngb improves mitochondria respiration, reduces ROS and RNS formation, inhibits cell death signaling, and diminishes glutamate release processes that modulate survival. In Aim 1, we will examine the role of Ngb in regulating mitochondria function and oxidative stress- mediated neuronal death after oxygen-glucose deprivation and hypoxic insults in vitro. Biomarkers of mitochondria integrity, ROS, and NO/ RNS production, cell death signaling, and cytotoxicity will be examined. Responses in Ngb over-expressing neurons will be compared with control (wild-type) mouse cortical neurons, with special attention to baseline levels in the Ngb transgenic neurons. In Aim 2, we will assess neuroprotective effects of Ngb in cerebral ischemia in vivo. Profiles of Ngb expression will be examined after transient (2 hrs) focal cerebral ischemia in mice by western blot, immunohistochemistry and RT-PCR. Mitochondria ATP levels, ROS and RNS production, cell death signaling, glutamate release will be examined. All measurements of baselines and changes after stroke will be compared in Ngb over-expressing transgenic mice versus wild-type littermates. In Aim 3, we will investigate roles of Ngb in neurological outcome and functional recovery after cerebral ischemia in vivo. After acute focal stroke for 1-6 hrs, clinically relevant surrogate markers of energetic stress will be analyzed by MR imaging. At 3 days, morphological outcomes including infarction, edema and BBS leakage will be measured. A standard battery of tests will be used to assess neurological recovery. Baselines and changes of endpoints for Ngb over-expressing transgenic mice will be compared against matching wild-type littermates after permanent and transient 2 hr focal cerebral ischemia. These proposed experiments should provide new insight into how Ngb protects neurons from hypoxia/ischemia and may ultimately lead to novel therapeutic strategies for the acute treatment of stroke.